5632

879

264

4506 18226

Ear2 Ear1

Tassel1+Tassel2

Figure S1: Number of peaks identified by KN1 ChIP-seq in the four

biological replicates. A total of 6511 peaks, referred as high

confidence-bound loci, overlap between the two ears samples.

C

KN1 - + - + - + - + - +

0

2

4

6

8

10

1 2 3 4 5 6 7 8 9 10En

ric

hm

en

t re

lati

ve

to

tu

b

500 bp

A

0

20

40

60

80

100

+/- -/- +/- -/- a b c d e f

a b c d e f

lg3

Ear1

Ear2

Primers *

B73 Ears SAM Tassels

B

EMSA

ChIP-seq

ChIP-qPCR

Figure S2: ChIP-seq results and validation. (A) ChIP-seq identified KN1 binding in liguleless3 (lg3). Genic exon-intron

structures is shown with red arrow indicating gene orientation. The blue bars with letters a-f mark primer positions used for

qPCR in (B). Longer bars mark positions of probes used for EMSA in (C), while the star (*) shows the position of a short probe

used for EMSA (Supplemental Fig. 6). (B) ChIP-qPCR validation of KN1 binding at the lg3 locus. Primers in position “c” were

used for ChIP-qPCR in SAM and tassels (right). Enrichment is not detected in kn1-e1 (-/-). Error bars correspond to the

standard variation of two (SAM and tassels) or three (ears) biological replicates. (C) EMSA using a 1 kb probe (black bar) or

shorter overlapping probes (colored bars) from lg3 (A). Of the short probes, the light blue and pink probes generated the

strongest binding (colored arrowheads), consistent with the binding to a motif present in the overlap of the two probes [star (*)

in (A), “lg3” probe in Supplemental Fig. 6C)]. The weak binding detected with the red and green probes is occasionally

observed in regions not bound in vivo and considered as non-specific.

0

10

20

30

40

50

60

70

80

90

NB

149

_N

B15

0

NB

502

_N

B50

3

NB

434

_N

B43

5

NB

793

_N

B79

4

NB

498

_N

B49

9

NB

735

_N

B73

6

NB

781

_N

B78

2

KM

10

2_K

M1

03

KM

10

4_K

M1

05

KM

10

6_K

M1

07

KM

10

8_K

M1

09

KM

11

0_K

M1

11

KM

11

2_K

M1

13

KM

11

4_K

M1

15

KM

11

6_K

M1

17

KM

86

_K

M87

KM

88

_K

M89

KM

90

_K

M91

KM

92

_K

M93

KM

94

_K

M95

KM

96

_K

M97

KM

98

_K

M99

NB

366

_N

B36

7

NB

370

_N

B37

1

NB

376

_N

B37

7

NB

382

_N

B38

3

NB

396

_N

B39

7

NB

406

_N

B40

7

NB

414

_N

B41

5

NB

432

_N

B43

3

NB

442

_N

B44

3

NB

448

_N

B44

9

NB

454

_N

B45

5

NB

472

_N

B47

3

NB

504

_N

B50

5

NB

506

_N

B50

7

NB

747

_N

B74

8

NB

769

_N

B77

0

NB

787

_N

B78

8

NB

795

_N

B79

6

En

ric

hm

en

t re

lati

ve

to

tu

bu

lin

Ears WT

negatives

Figure S3: ChIP-qPCR validation in wild-type ears. Primer pairs labeled “negatives” are those called negative in all three wild-

type tissues tested in Supplemental Table 3. Errors bars are the standard variation of two biological replicates.

0

50

100

150

200

250

NB

149

_N

B15

0

NB

502

_N

B50

3

NB

434

_N

B43

5

NB

793

_N

B79

4

NB

498

_N

B49

9

KM

10

2_K

M1

03

KM

10

4_K

M1

05

KM

10

6_K

M1

07

KM

10

8_K

M1

09

KM

11

0_K

M1

11

KM

11

2_K

M1

13

KM

11

4_K

M1

15

KM

11

6_K

M1

17

KM

86

_K

M87

KM

88

_K

M89

KM

90

_K

M91

KM

92

_K

M93

KM

94

_K

M95

KM

96

_K

M97

KM

98

_K

M99

NB

366

_N

B36

7

NB

370

_N

B37

1

NB

376

_N

B37

7

NB

382

_N

B38

3

NB

396

_N

B39

7

NB

406

_N

B40

7

NB

414

_N

B41

5

NB

432

_N

B43

3

NB

442

_N

B44

3

NB

448

_N

B44

9

NB

454

_N

B45

5

NB

472

_N

B47

3

NB

504

_N

B50

5

NB

506

_N

B50

7

NB

747

_N

B74

8

NB

769

_N

B77

0

NB

795

_N

B79

6

En

ric

hm

en

t re

lati

ve

to

tu

bu

lin

Tassels WT

Tassels kn1-e1

negatives

Figure S4: ChIP-qPCR validation in wild-type (WT) and kn1-e1 tassels. Primer pairs labeled “negatives” are those called negative

in all three wild-type tissues tested in Supplemental Table 3. Errors bars are the standard variation of two biological replicates.

0

20

40

60

80

100

120

140

160

180

200

NB

149

_N

B15

0

NB

502

_N

B50

3

NB

434

_N

B43

5

NB

793

_N

B79

4

NB

498

_N

B49

9

KM

10

2_K

M1

03

KM

10

4_K

M1

05

KM

10

6_K

M1

07

KM

10

8_K

M1

09

KM

11

0_K

M1

11

KM

11

2_K

M1

13

KM

11

4_K

M1

15

KM

11

6_K

M1

17

KM

86

_K

M87

KM

88

_K

M89

KM

90

_K

M91

KM

92

_K

M93

KM

94

_K

M95

KM

96

_K

M97

KM

98

_K

M99

NB

366

_N

B36

7

NB

370

_N

B37

1

NB

376

_N

B37

7

NB

382

_N

B38

3

NB

396

_N

B39

7

NB

406

_N

B40

7

NB

414

_N

B41

5

NB

432

_N

B43

3

NB

442

_N

B44

3

NB

448

_N

B44

9

NB

454

_N

B45

5

NB

472

_N

B47

3

NB

504

_N

B50

5

NB

506

_N

B50

7

NB

747

_N

B74

8

NB

769

_N

B77

0

NB

795

_N

B79

6

KM

10

0_K

M1

01

En

ric

hm

en

t re

lati

ve

to

tu

bu

lin

SAM WT

SAM kn1-e1

negatives

Figure S5: ChIP-qPCR validation in wild-type (WT) and kn1-e1 SAM. Primer pairs labeled “negatives” are those called negative in

all three wild-type tissues tested in Supplemental Table 3. Errors bars are the standard variation of two biological replicates.

GA

P0

G

AP

1

GA

P2

G

AP

2b

G

AP

3

GA

P3

b

W

T

GA

P3

G

AP

3b

G

AP

3c

WT

WT

V

1

V2

V

3

V4

B

Bound

probe

Free

probe

MD1

MD4 MD3

MD2

A

WT

C

1/2

M

D1a

M

D1b

M

D2a

M

D2

b

MD

3a

M

D3b

M

D4

Free

probe

Bound

probe

C

ga2o

x1

lg3

ga-c

1

ga-c

1/2

kn

1-1

tb1

lg3

Bound

probe

Free

probe

ga2ox1 TGACTGATTGAC

lg3 TGATGATATGAT

ga-c1 TCGCTGATTGAC

ga-c1/2 TCGCTGATTCGC

kn1-1 TGACAGATAGAC

tb1 TGATGGATGGAC

Figure S6: see next page for legend

Figure S6: KN1 binds in vitro to motifs identified in the in vivo KN1-bound sequences. (A) We carried out motif

discovery starting from the high confidence KN1-bound regions. Motifs were identified that appear as two NGAC

sequences spaced by one or two residues, instead of the four residues found in the ga2ox1 binding site

(Supplemental Table 4). However, no KN1 binding was detected through EMSA assay using oligos derived from

these motifs (right). (B) EMSA comparing KN1 binding to the ga2ox1 binding site (labeled as “WT”) with the binding

to variants of the WT motif. (C) EMSA comparing KN1 in vitro binding to motifs derived from ga2ox1, lg3, kn1 and

tb1 (see Supplemental Table 4 for full probes sequences). The weak ga2ox1 variant ga-c1 could recruit KN1 in

planta as efficiently as the original ga2ox1 motif, suggesting that weaker motifs such as lg3 are sufficient to promote

KN1 binding in vivo.

0.E+00

1.E-05

2.E-05

3.E-05

4.E-05

5.E-05

up 10 kb up 1 kb 5'UTR exon intron 3'UTR down 1 kb down 10 kb

Pe

ak

de

ns

ity (

pe

ak

nu

mb

er/

bp

)

Figure S7: Detailed distribution of KN1 peaks around transcribed genes. Because exons, introns and UTRs vary in size,

the number of peaks was normalized relative to the size (bp) of each gene structure. The up/down 1 and 10 kb represent

all the peaks located from the UTR to 1 or 10 kb away, respectively. Thus, the “10 kb” categories also include the peaks in

the “1 kb” categories.

- + KN1 C

3000 bp

64 kb

tb1

Ear1

Ear2

*

A

*

B

-66 kb -65 kb -64 kb -63 kb -62 kb

Tourist Hopscotch

408 bp 4,885 bp

* TGATGGATGGAC

ChIP-seq

Figure S8: KN1 binding at the tb1 locus. (A) A binding site is detected 64 kb upstream of the tb1 transcription start, within

the region previously identified as responsible for morphological differences between maize and teosinte (58-69 kb

upstream of the transcription start; Clark et al 2006). The red bar marks the region used for the EMSA in (C). The star (*)

shows the position of the oligo probe used for the EMSA in Fig. S6C. Another peak is located 10 kb downstream of the

transcription end, in addition to a peak detected only in Ear1 in the 5’ of the gene. (B) KN1 binding site (red star) similar to

that found in ga2ox1 was identified immediately upstream of the Hopscotch transposon found to be important for tb1

expression (Studer et al 2011). Positions are relative to the transcription start site of tb1. (B) EMSA identifying KN1

binding in vitro (arrow) to the same region bound in vivo.

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

kn1 ga2ox1 lg3

Rela

tive

ex

pre

ss

ion

+/+

Kn1-N/+

Kn1-N/Kn1-N

Figure S9: Quantification of kn1, ga2ox1 and lg3 mRNA accumulation in leaf primordia from wild-type

(+/+) and Kn1-N seedlings. ga2ox1 and lg3 mRNA levels in Kn1-N leaf primordia follow a gradient of

expression proportional to kn1 overexpression. Error bars are the standard deviation of two biological

replicates.

+/+ Kn1-N/+ Kn1-N/Kn1-N

A B

2G087741

2G094241

2G452178

2G135447

2G102161

2G126018

2G074543

2G167824

2G005353

2G529859

2G116646

2G079080

2G021406

lg3

lg4a

gn1

knox8

zmm8

tsh4

yab9

yab10

yab14

yab15

yab5

ago

log

FDR < 5E-2 *

< 1E-2 **

< 1E-4 ***

< 1E-8 ****

-2.0

-1.75

-1.5

-1.1

1.0

1.1

1.25

1.5

2.0

20

40

♀ ♂ K/+ K/K GRMZM

0

0.01

0.02

0.03

0.04

0.05

Flu

ore

sc

en

ce

/ug

to

tal

pro

tein

+/+

Kn

1-N

/+

+/+

; D

R5

Kn

1-N

/+;

DR

5

*

C

argonaute GRMZM2G079080

E1

E2

E1

E2

1 kb

2 kb

lonely guy GRMZM2G021406 D

Figure S10: (A) Kn1-N phenotype. Shown are 10 days old seedlings, the stage used for sequencing. (B) Changes in

gene expression for selected direct targets in ears (♀), tassels (♂) or leaves [Kn1-N heterozygotes (K/+) and

homozygotes (K/K)]. (C) Quantification of fluorescence in protein extracts from immature leaves of Kn1-N/+ plants and

wild-type siblings segregating the DR5rev::mRFPer reporter (DR5). The difference between +/+;DR5 and Kn1-N/+;DR5

is statistically significant (*; P-value = 0.0003). Error bars correspond to the standard variation of 9 (non-DR5 siblings)

or 18 (DR5) biological replicates. (D) ChIP-seq identified KN1 binding in selected targets. Exon-intron structure is

shown with red arrows indicating gene orientation. Peak traces are shown for the two biological replicates of ears (E1-

E2).

bHLH GRMZM2G080054

Ear1

Ear2

5 kb

Figure S11: Peaks detected around gene GRMZM2G080054 (bHLH), which is up-regulated in ears (fold change

3.07, P value = 1.8E-06). Other genes in the vicinity are either not expressed, or expressed at a low level and

are not modulated.

MEME version 4

ALPHABET= ACGT

strands: + -

Background letter frequencies

A 0.275 C 0.225 G 0.225 T 0.275

MOTIF ga2ox1 like

letter-probability matrix: alength= 4 w= 12

0.000000 0.000000 0.000000 1.000000

0.000000 0.000000 1.000000 0.000000

1.000000 0.000000 0.000000 0.000000

0.000000 0.820000 0.000000 0.180000

0.100000 0.080000 0.320000 0.500000

0.000000 0.000000 1.000000 0.000000

1.000000 0.000000 0.000000 0.000000

0.000000 0.380000 0.000000 0.620000

0.250000 0.000000 0.250000 0.500000

0.000000 0.000000 1.000000 0.000000

1.000000 0.000000 0.000000 0.000000

0.000000 0.820000 0.000000 0.180000

Figure S12: Probability matrix used to identify KN1 binding sites in the KN1-bound loci and B73

genome.